Synthetic mammalian α-N-acetylglucosaminidase and genetic sequences encoding same

ABSTRACT

The present invention relates generally to mammalian α-N-acetyglucosaminidase and to genetic sequences encoding same and to their use in the investigation, diagnosis and treatment of subjects suspected of or suffering from α-N-acetyglucosaminidase deficiency.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. Ser. No.09/836,613, filed Apr. 17, 2001 now abandoned, which is a divisionalapplication of U.S. Ser. No. 09/077,354 now U.S. Pat. No. 6,255,096,which was a section 371 application of PCT/AU96/00747 having anInternational Filing Date of Nov. 22, 1996.

FIELD OF THE INVENTION

The present invention relates generally to mammalianα-N-acetylglucosaminidase and to genetic sequences encoding same and tothe use of these in the investigation, diagnosis and treatment ofsubjects suspected of or suffering from α-N-acetylglucosaminidasedeficiency.

Bibliographic details of the publications referred to by author in thisspecification are collected at the end of the description. SequenceIdentity Numbers (SEQ ID NOs.) for the nucleotide and amino acidsequences referred to in the specification are defined following thebibliography.

Throughout this specification, unless the context requires otherwise,the word “comprise”, or variations such as “comprises” or “comprising”,will be understood to imply the inclusion of a stated element or integeror group of elements or integers but not the exclusion of any otherelement or integer or group of elements or integers.

BACKGROUND TO THE INVENTION

The increasing sophistication of recombinant DNA technology is greatlyfacilitating the efficacy of many commercially important industriesincluding areas of medical and pharmaceutical research and development.The ability to purify native proteins and subsequently clone geneticsequences encoding these proteins is an important first step in thedevelopment of a range of therapeutic and diagnostic procedures.However, practitioners have faced many difficulties in purifying targetmolecules to an extent sufficient to determine amino acid sequences topermit the development of oligonucleotide probes to assist in thecloning of genetic sequences encoding the target molecules.

Such difficulties have been particularly faced in the research anddevelopment of lysosomal enzymes. An important lysosomal enzyme isα-N-acetylglucosaminidase (EC 2.1.50). This enzyme acts as aexoglycosidase in lysosomes to hydrolyse the terminalα-N-acetylglucosamine residues present at the non-reducing terminus offragments of heparan sulphate and heparin (Hopwood, 1989). A deficiencyin this lysosomal hydrolase is responsible for the pathogenesis ofSanfilippo B (Mucopolysaccharidosis type IIIB [MPS-IIIB]) syndrome(von-Figura and Kresse, 1972; O'Brien, 1972). This is an autosomalrecessive disorder of glycosaminoglycan catabolism leading to storageand excretion of excessive amounts of heparan sulphate and a variety ofclinical phenotypes, but classically presenting with progressive mentalretardation in conjunction with skeletal deformities (McKusick andNeufeld, 1983).

There is a need, therefore, to purify α-N-acetylglucosaminidase and toclone genetic sequences encoding same to permit development of a rangeof therapeutic and diagnostic procedures to assist in the diagnosis andtreatment of disease conditions arising from α-N-acetylglucosaminidasedeficiency.

SUMMARY OF THE INVENTION

One aspect of the invention provides an isolated nucleic acid moleculecomprising a sequence of nucleotides which encodes or is complementaryto a sequence which encodes a mammalian α-N-acetylglucosaminidase orfragment or derivative thereof.

A second aspect of the invention provides an isolated nucleic acidmolecule comprising a sequence of nucleotides which is capable ofhybridising under at least low stringency conditions to a nucleotidesequence set forth in SEQ ID NO:1 or SEQ ID NO:3 or a complementarystrand or a homologue, analogue or derivative thereof.

Another aspect of the invention is directed an isolated nucleic acidmolecule which is at least 40% identical to the nucleotide sequence setforth in SEQ ID NO:1 or SEQ ID NO:3 or to a complementary strand thereofor a homologue, analogue or derivative thereof.

A further aspect of the present invention provides a nucleic acidmolecule comprising a sequence of nucleotides encoding or complementaryto a sequence encoding a polypeptide capable of hydrolysing the terminalα-N-acetylglucosamine residues present at the non-reducing terminus offragments of heparan sulphate and heparin residues and wherein saidnucleotide sequence is capable of hybridising under low stringencyconditions to the nucleotide sequence set forth in SEQ ID NO: 1.

A further aspect of the invention is directed to a genetic constructcomprising a sense molecule, for the expression or over-expression ofα-N-acetylglucosaminidase in prokaryotic or eukaryotic cells.

A further aspect of the present invention is directed to syntheticα-N-acetylglucosaminidase or like molecule.

A further aspect of the invention contemplates antibodies toα-N-acetylglucosaminidase and preferably syntheticα-N-acetylglucosaminidase or a like molecule.

In still yet another aspect of the present invention there iscontemplated a method of diagnosing a mutation or other abberations inthe α-N-acetylglucosaminidase gene in a human or animal patient.

Another aspect contemplates a method of treating patients suffering fromα-N-acetylglucosaminidase deficiency, such as in MPS-IIIB, said methodcomprising administering to said patient an effective amount ofα-N-acetylglucosaminidase or active like form thereof.

Another aspect of the present invention is directed to a pharmaceuticalcomposition comprising a recombinant mammalian α-N-acetylglucosaminidaseor an active fragment or derivative thereof and one or morepharmaceutically acceptable carriers and/or diluents.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a photographic representation of α-N-acetylglucosaminidasepurified from human placenta following SDS/PAGE. Lane 1: M_(r) standards(kDa); Lanes 2 and 3: purified α-N-acetylglucosaminidase from humanplacenta. Lane 4 and 5, bovine serum albumin.

FIG. 2 is a photographic representation of an SDS/polyacrylamide gelshowing the molecular weights of recombinant α-N-acetylglucosaminidasepolypeptides produced in CHO cells before (−) and after (+) PNGase Fdigestion. The 50 mM NaCl and 75 mM NaCl fractions are indicated.Molecular weights of α-N-acetylglucosaminidase polypeptides areindicated on the left of the figure. Molecular weights of markerproteins are indicated on the right hand side of the figure (lane 5).

Single and three letter abbreviations of conventional amino acidresidues as used herein are defined in Table 1.

Suitable amino acid substitutions referred to herein are defined inTable 2.

Codes for non-conventional amino acid residues as used herein aredefined in Table 3.

TABLE 1 Three-letter One-letter Amino Acid Abbreviation Symbol AlanineAla A Arginine Arg R Asparagine Asn N Aspartic acid Asp D Cysteine Cys CGlutamine Gln Q Glutamic acid Glu E Glycine Gly G Histidine His HIsoleucine Ile I Leucine Leu L Lysine Lys K Methionine Met MPhenylalanine Phe F Proline Pro P Serine Ser S Threonine Thr TTryptophan Trp W Tyrosine Tyr Y Valine Val V Any residue Xaa X

TABLE 2 Suitable residues for amino acid substitutions Original ResidueExemplary Substitutions Ala Ser Arg Lys Asn Gln; His Asp Glu Cys Ser GlnAsn Glu Asp Gly Pro His Asn; Gln Ile Leu; Val Leu Ile; Val Lys Arg; Gln;Glu Met Leu; Ile Phe Met; Leu; Tyr Ser Thr Thr Ser Trp Tyr Tyr Trp; PheVal Ile; Leu

TABLE 3 Non-conventional amino acid Code α-aminobutyric acid Abuα-amino-α-methylbutyrate Mgabu aminocyclopropane- Cpro carboxylateaminoisobutyric acid Aib aminonorbornyl- Norb carboxylatecyclohexylalanine Chexa cyclopentylalanine Cpen D-alanine Dal D-arginineDarg D-aspartic acid Dasp D-cysteine Dcys D-glutamine Dgln D-glutamicacid Dglu D-histidine Dhis D-isoleucine Dile D-leucine Dleu D-lysineDlys D-methionine Dmet D-ornithine Dorn D-phenylalanine Dphe D-prolineDpro D-serine Dser D-threonine Dthr D-tryptophan Dtrp D-tyrosine DtyrD-valine Dval D-α-methylalanine Dmala D-α-methylarginine DmargD-α-methylasparagine Dmasn D-α-methylaspartate Dmasp D-α-methylcysteineDmcys D-α-methylglutamine Dmgln D-α-methylhistidine DmhisD-α-methylisoleucine Dmile D-α-methylleucine Dmleu D-α-methyllysineDmlys D-α-methylmethionine Dmmet D-α-methylornithine DmornD-α-methylphenylalanine Dmphe D-α-methylproline Dmpro D-α-methylserineDmser D-α-methylthreonine Dmthr D-α-methyltryptophan DmtrpD-α-methyltyrosine Dmty D-α-methylvaline Dmval D-N-methylalanine DnmalaD-N-methylarginine Dnmarg D-N-methylasparagine DnmasnD-N-methylaspartate Dnmasp D-N-methylcysteine Dnmcys D-N-methylglutamineDnmgln D-N-methylglutamate Dnmglu D-N-methylhistidine DnmhisD-N-methylisoleucine Dnmile D-N-methylleucine Dnmleu D-N-methyllysineDnmlys N-methylcyclohexylalanine Nmchexa D-N-methylornithine DnmornN-methylglycine Nala N-methylaminoisobutyrate NmaibN-(1-methylpropyl)glycine Nile N-(2-methylpropyl)glycine NleuD-N-methyltryptophan Dnmtrp D-N-methyltyrosine Dnmtyr D-N-methylvalineDnmval γ-aminobutyric acid Gabu L-t-butylglycine Tbug L-ethylglycine EtgL-homophenylalanine Hphe L-α-methylarginine Marg L-α-methylaspartateMasp L-α-methylcysteine Mcys L-α-methylglutamine MglnL-α-methylhistidine Mhis L-α-methylisoleucine Mile L-α-methylleucineMleu L-α-methylmethionine Mmet L-α-methylnorvaline MnvaL-α-methylphenylalanine Mphe L-α-methylserine Mser L-α-methyltryptophanMtrp L-α-methylvaline Mval N-(N-(2,2-diphenylethyl) Nnbhmcarbamylmethyl)glycine 1-carboxy-1-(2,2-diphenyl- Nmbcethylamino)cyclopropane L-N-methylalanine Nmala L-N-methylarginine NmargL-N-methylasparagine Nmasn L-N-methylaspartic acid NmaspL-N-methylcysteine Nmcys L-N-methylglutamine Nmgln L-N-methylglutamicacid Nmglu L-N-methylhistidine Nmhis L-N-methylisolleucine NmileL-N-methylleucine Nmleu L-N-methyllysine Nmlys L-N-methylmethionineNmmet L-N-methylnorleucine Nmnle L-N-methylnorvaline NmnvaL-N-methylornithine Nmorn L-N-methylphenylalanine NmpheL-N-methylproline Nmpro L-N-methylserine Nmser L-N-methylthreonine NmthrL-N-methyltryptophan Nmtrp L-N-methyltyrosine Nmtyr L-N-methylvalineNmval L-N-methylethylglycine Nmetg L-N-methyl-t-butylglycine NmtbugL-norleucine Nle L-norvaline Nva α-methyl-aminoisobutyrate Maibα-methyl-γ-aminobutyrate Mgabu α-methylcyclohexylalanine Mchexaα-methylcylcopentylalanine Mcpen α-methyl-α-napthylalanine Manapα-methylpenicillamine Mpen N-(4-aminobutyl)glycine NgluN-(2-aminoethyl)glycine Naeg N-(3-aminopropyl)glycine NornN-amino-α-methylbutyrate Nmaabu α-napthylalanine Anap N-benzylglycineNphe N-(2-carbamylethyl)glycine Ngln N-(carbamylmethyl)glycine NasnN-(2-carboxyethyl)glycine Nglu N-(carboxymethyl)glycine NaspN-cyclobutylglycine Ncbut N-cycloheptylglycine Nchep N-cyclohexylglycineNchex N-cyclodecylglycine Ncdec N-cylcododecylglycine NcdodN-cyclooctylglycine Ncoct N-cyclopropylglycine NcproN-cycloundecylglycine Ncund N-(2,2-diphenylethyl)glycine NbhmN-(3,3-diphenylpropyl)glycine Nbhe N-(3-guanidinopropyl)glycine NargN-(1-hydroxyethyl)glycine Nthr N-(hydroxyethyl))glycine NserN-(imidazolylethyl))glycine Nhis N-(3-indolylyethyl)glycine NhtrpN-methyl-γ-aminobutyrate Nmgabu D-N-methylmethionine DnmmetN-methylcyclopentylalanine Nmcpen D-N-methylphenylalanine DnmpheD-N-methylproline Dnmpro D-N-methylserine Dnmser D-N-methylthreonineDnmthr N-(1-methylethyl)glycine Nval N-methyla-napthylalanine NmanapN-methylpenicillamine Nmpen N-(p-hydroxyphenyl)glycine NhtyrN-(thiomethyl)glycine Ncys penicillamine Pen L-α-methylalanine MalaL-α-methylasparagine Masn L-α-methyl-t-butylglycine MtbugL-methylethylglycine Metg L-α-methylglutamate Mglu L-α-methylhomophenylalanine Mhphe N-(2-methylthioethyl)glycine Nmet L-α-methyllysineMlys L-α-methylnorleucine Mnle L-α-methylornithine MornL-α-methylproline Mpro L-α-methylthreonine Mthr L-α-methyltyrosine MtyrL-N-methylhomo phenylalanine Nmhphe N-(N-(3,3-diphenylpropyl) Nnbhecarbamylmethyl)glycine

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention provides an isolated nucleic acid moleculecomprising a sequence of nucleotides which encodes, or are complementaryto a sequence which encodes, a mammalian α-N-acetylglucosaminidase orfragment or derivative thereof or its like molecule.

Preferably, the mammal is a human, livestock animal, companion animal,wild animal or laboratory test animal (e.g. rabbit, rat, mouse or guineapig). Most preferably, the mammal is a human. Conveniently, theα-N-acetylglucosaminidase is isolatable from the liver, kidney orplacenta. However, the present invention extends to all mammalianα-N-acetylglucosaminidase enzymes and from any anatomical or cellularsource and/or any biological fluid source, such as but not limited toplasma, serum, cell extract or lymph fluid.

Although a preferred embodiment of the present invention contemplatesthe use of human α-N-acetylglucosaminidase or genomic or recombinant(e.g. cDNA) genetic sequences encoding same in the investigation,diagnosis and/or treatment of human subjects (i.e. homologous system),one skilled in the art will appreciate that the enzyme or geneticsequences encoding same from a non-human animal may also be useful. Sucha heterologous system is encompassed by the present invention.

The term “nucleic acid molecule” as used herein shall be taken to referto any RNA or DNA (e.g. cDNA) molecule, whether single-stranded ordouble-stranded or in a linear or covalently-closed form. The nucleicacid molecule may also be DNA corresponding to the entire genomic geneor a substantial portion thereof or a fragment or derivative thereof.

The nucleic acid molecule of the present invention may constitute solelythe nucleotide sequence encoding α-N-acetylglucosaminidase or aα-N-acetylglucosaminidase-like molecule or may be part of a largernucleic acid molecule. Accordingly, the present invention extends to theisolated genomic α-N-acetylglucosaminidase gene. The non-translatedsequences in a larger nucleic acid molecule may include vector,transcriptional and/or translational regulatory sequences, promoter,terminator, enhancer, replication or signal sequences or non-codingregions (eg intron sequences) of an isolated genomic gene.

Reference herein to a “gene” is to be taken in its broadest context andincludes:

-   -   (i) a classical genomic gene consisting of transcriptional        and/or translational regulatory sequences and/or a coding region        and/or non-translated sequences (i.e. introns, 5′- and        3′-untranslated sequences);    -   (ii) mRNA or cDNA corresponding to the coding regions (i.e.        exons) optionally comprising 5′- or 3′-untranslated sequences of        the gene; or    -   (iii) synthetic, amplified DNA fragments or other recombinant        nucleic acid molecules produced in vitro and comprising all or a        part of the coding region and/or 5′- or 3′-untranslated        sequences of the gene.

The term “gene” is also used to describe synthetic or fusion moleculesencoding all or part of a functional product. A functional product isone which comprises a sequence of nucleotides or is complementary to asequence of nucleotides which encodes a functional polypeptide, inparticular a polypeptide having the catalytic activity ofα-N-acetylglucosaminidase or a homologue, analogue or derivativethereof.

For the present purpose, “homologues” of a nucleotide sequence shall betaken to refer to an isolated nucleic acid molecule which issubstantially the same as the nucleic acid molecule of the presentinvention or its complementary nucleotide sequence, notwithstanding theoccurrence within said sequence, of one or more nucleotidesubstitutions, insertions, deletions, or rearrangements.

“Analogues” of a nucleotide sequence set forth herein shall be taken torefer to an isolated nucleic acid molecule which is substantially thesame as a nucleic acid molecule of the present invention or itscomplementary nucleotide sequence, notwithstanding the occurrence of anynon-nucleotide constituents not normally present in said isolatednucleic acid molecule, for example carbohydrates, radiochemicalsincluding radionucleotides, reporter molecules such as, but not limitedto DIG, alkaline phosphatase or horseradish peroxidase, amongst others.

“Derivatives” of a nucleotide sequence set forth herein shall be takento refer to any isolated nucleic acid molecule which containssignificant sequence similarity to said sequence or a part thereof.Generally, the nucleotide sequence of the present invention may besubjected to mutagenesis to produce single or multiple nucleotidesubstitutions, deletions and/or insertions. Nucleotide insertionalderivatives of the nucleotide sequence of the present invention include5′ and 3′ terminal fusions as well as intra-sequence insertions ofsingle or multiple nucleotides or nucleotide analogues. Insertionalnucleotide sequence variants are those in which one or more nucleotidesor nucleotide analogues are introduced into a predetermined site in thenucleotide sequence of said sequence, although random insertion is alsopossible with suitable screening of the resulting product beingperformed. Deletional variants are characterised by the removal of oneor more nucleotides from the nucleotide sequence. Substitutionalnucleotide variants are those in which at least one nucleotide in thesequence has been removed and a different nucleotide or nucleotideanalogue inserted in its place.

Preferably, a homologue, analogue or derivative of anα-N-acetylglucosaminidase gene according to any embodiments describedherein, comprises a sequence of nucleotides of at least 10 contiguousnucleotides derived from SEQ ID NO:1 or SEQ ID NO:3 or a complementarystrand thereof, wherein the sequence of said homologue, analogue orderivative is at least 40% identical to SEQ ID NO:1 or SEQ ID NO:3 or acomplementary strand thereof or wherein said homologue, analogue orderivative is capable of hybridising to said sequence under at least lowstringency hybridisation conditions.

For the purposes of nomenclature, the nucleotide sequence set for in SEQID NO: 1 relates to the cDNA encoding the humanα-N-acetylglucosaminidase enzyme.

The nucleotide sequence set forth in SEQ ID NO:3 relates to the genomicgene equivalent of the cDNA encoding the human liverα-N-acetylglucosaminidase enzyme. Those skilled in the art will be awarethat the specific exon sequences described in SEQ ID NO:3 correspond tothe coding regions of the α-N-acetylglucosaminidase gene, said exonregions further comprising the entire open reading frame of the cDNAsequence set forth in SEQ ID NO:1, when aligned in a head-to-tailconfiguration. The intron sequences of SEQ ID NO:3, which correspond tonon-coding regions of the gene which are spliced from the primarytranscription product thereof, although not explicitly defined, may bereadily deduced by those skilled in the art, when provided with the exonsequence data provided in the nucleotide sequence listing.

The nucleotide sequence of the present invention may correspond to thesequence of the naturally-occurring α-N-acetylglucosaminidase gene ormay comprise a homologue, analogue or derivative thereof which containssingle or multiple nucleotide substitutions, deletions and/or additions.All such homologues, analogue or derivatives encodeα-N-acetylglucosaminidase or α-N-acetylglucosaminidase-like molecules ora homologue, analogue or derivative thereof as contemplated by thepresent invention. The length of the nucleotide sequence may vary from afew bases, such as in nucleic acid probes or primers, to a full lengthsequence.

The present invention is particularly directed to the nucleic acid incDNA form and particularly when inserted into an expression vector. Theexpression vector may be replicable in a eukaryotic or prokaryotic celland may either produce mRNA or the mRNA may be subsequently translatedinto α-N-acetylglucosaminidase or like molecule. Particularly preferredeukaryotic cells include CHO cells but may be in any other suitablemammalian cells or cell lines or non-mammalian cells such as yeast orinsect cells.

In an alternative embodiment, the present invention provides a nucleicacid molecule comprising a sequence of nucleotides which encodes or arecomplementary to a sequence which encodes a polypeptide capable ofhydrolysing the α-N-acetylglucosamine residues from the non-reducingterminus of heparan sulphate and heparin fragments and wherein saidnucleotide sequence is capable of hybridising under at least lowstringency conditions to a nucleotide sequence set forth in SEQ ID NO: 1or SEQ ID NO:3 or a homologue, analogue or derivative thereof.

A second aspect of the invention provides an isolated nucleic acidmolecule comprising a sequence of nucleotides which is capable ofhybridising under at least low stringency conditions to a nucleotidesequence set forth in SEQ ID NO:1 or SEQ ID NO:3 or a complementarystrand or a homologue, analogue or derivative thereof.

Preferably, hybridisation is possible under at least medium stringentconditions. More preferably, hybridisation is possible under highstringent conditions.

For the purposes of defining the level of stringency, reference canconveniently be made to Sambrook et al (1989) or Ausubel et al (1987)which are herein incorporated by reference.

A low stringency is defined herein as being a hybridisation and/or washcarried out in 4-6×SSC/0.1-0.5% w/v SDS at 37-45° C. for 2-3 hours. Amedium stringency hybridisation and/or wash is carried out in1-4×SSC/0.25-0.5% w/v SDS at ≧45° C. for 2-3 hours and a high stringencyhybridisation and/or wash is carried out 0.1-1×SSC/0.1% w/v SDS at 60°C. for 1-3 hours.

Alternative conditions of stringency may be employed to thosespecifically recited herein. Generally, the stringency is increased byreducing the concentration of SSC buffer, and/or increasing theconcentration of SDS and/or increasing the temperature of thehybridisation and/or wash. Those skilled in the art will be aware thatthe conditions for hybridisation and/or wash may vary depending upon thenature of the hybridisation membrane or the type of hybridisation probeused. Conditions for hybridisations and washes are well understood byone normally skilled in the art. For the purposes of clarification ofparameters affecting hybridisation between nucleic acid molecules,reference is found in pages 2.10.8 to 2.10.16. of Ausubel et al. (1987),which is herein incorporated by reference.

Those skilled in the art will be aware that the nucleotide sequences setforth in SEQ ID NO: 1 and SEQ ID NO:3 may be used to isolate thecorresponding genes from other human tissues or alternatively, from thetissues or cells of other species, without undue experimentation. Meansfor the isolated of such related sequences will also be known to thoseskilled in the art, for example nucleic acid hybridisation, polymerasechain reaction, antibody screening of expression libraries, functionalscreening of expression libraries, or complementation of mutants,amongst others. The present invention is not to be limited by the sourcefrom which the specific gene sequences described herein have beenisolated or by the means used to isolate said sequences.

In one embodiment, a related genetic sequence comprising genomic DNA, ormRNA, or cDNA is contacted with a hybridisation effective amount of agenetic sequence which encodes α-N-acetylglucosaminidase, or itscomplementary nucleotide sequence or a homologue, analogue, derivativeor functional part thereof, and then said hybridisation is detectedusing a suitable detection means.

The related genetic sequence may be in a recombinant form, in a virusparticle, bacteriophage particle, yeast cell, animal cell, or a plantcell. Preferably, the related genetic sequence originates from an animalspecies or a human. More preferably, the related genetic sequenceoriginates from a human.

Preferably, the genetic sequence which encodes α-N-acetylglucosaminidase(i.e. probe or latter genetic sequence) comprises a sequence ofnucleotides of at least 10 nucleotides, more preferably at least 20nucleotides, even more preferably at least 50 nucleotides and even stillmore preferably at least 100 nucleotides derived from the nucleotidesequence set forth in SEQ ID NO:1 or SEQ ID NO:3 or a complementarysequence or a homologue, analogue or derivative thereof.

Preferably, the detection means is a reporter molecule capable of givingan identifiable signal (e.g. a radioisotope such as ³²P or ³⁵S or abiotinylated molecule) covalently attached to theα-N-acetylglucosaminidase probe.

In an alternative embodiment, the detection means is a polymerase chainreaction. According to this embodiment, two opposing non-complementarynucleic acid “primer molecules” of at least 10 nucleotides in length,more preferably at least 20 nucleotides in length, derived from thenucleotide sequence set forth in SEQ ID NO:1 or SEQ ID NO:3 may becontacted with a nucleic acid “template molecule” and specific nucleicacid molecule copies of the template molecule amplified in a polymerasechain reaction.

The opposing primer molecules are selected such that they are eachcapable of hybridising to complementary strands of the same templatemolecule, wherein DNA polymerase-dependant DNA synthesis occurring froma first opposing primer molecule will be in a direction toward thesecond opposing primer molecule.

Accordingly, both primers hybridise to said template molecule such that,in the presence of a DNA polymerase enzyme, a cofactor and appropriatesubstrate, DNA synthesis occurs in the 5′ to 3′ direction from eachprimer molecule towards the position on the DNA where the other primermolecule is hybridised, thereby amplifying the intervening DNA.

Those skilled in the art are aware of the technical requirements of thepolymerase chain reaction and are capable of any modifications which maybe made to the reaction conditions. For example, of the polymerase chainreaction may be used in any suitable format, such as amplified fragmentlength polymorphism (AFLP), single-strand chain polymorphism (SSCP),amplification and mismatch detection (AMD), interspersed repetitivesequence polymerase chain reaction (IRS-PCR), inverse polymerase chainreaction (iPCR) and reverse transcription polymerase chain reaction(RT-PCR), amongst others, to isolate a related α-N-acetylglucosaminidasegene sequence or identify a mutation in an α-N-acetylglucosaminidasegenetic sequence. Such variations of the polymerase chain reaction arediscussed in detail by McPherson et al (1991), which is incorporatedherein by reference. The present invention encompasses all suchvariations, the only requirement being that the final product of thereaction is an isolated nucleic acid molecule which is capable ofencoding α-N-acetylglucosaminidase or a homologue, analogue orderivative thereof.

In a preferred embodiment, the first primer molecule is preferablyderived from the sense strand of a gene which encodesα-N-acetylglucosaminidase, in particular from the nucleotide sequenceset forth in SEQ ID NO: 1 or SEQ ID NO:3 or a homologue, derivative oranalogue thereof and the second primer molecule is preferably derivedfrom the antisense strand of said gene.

Those skilled in the art will be aware that it is not essential to theperformance of the invention that the primer molecules be derived fromthe same gene.

According to this embodiment, the nucleic acid primer molecule mayfurther consist of a combination of any of the nucleotides adenine,cytidine, guanine, thymidine, or inosine, or functional analogues orderivatives thereof, capable of being incorporated into a polynucleotidemolecule provided that it is capable of hybridising under at least lowstringency conditions to the nucleic acid molecule set forth in SEQ IDNO:1 or SEQ ID NO:3 or a homologue, analogue or derivative thereof.

The nucleic acid primer molecules may further be each contained in anaqueous pool comprising other nucleic acid primer molecules. Morepreferably, the nucleic acid primer molecule is in a substantially pureform.

The nucleic acid template molecule may be in a recombinant form, in avirus particle, bacteriophage particle, yeast cell, animal cell, or aplant cell. Preferably, the related genetic sequence originates from acell, tissue, or organ derived from an animal species or a human. Morepreferably, the related genetic sequence originates from a cell, tissue,or organ derived from a human.

Accordingly, a third aspect of the present invention extends to anisolated nucleic acid molecule which is at least 40% identical to thenucleotide sequence set forth in SEQ ID NO:1 or SEQ ID NO:3 or to acomplementary strand thereof or a homologue, analogue or derivativethereof.

Preferably, the percentage identity to SEQ ID NO:1 or SEQ ID NO:3 is atleast about 55%, still more preferably at least about 65%, yet stillmore preferably at least about 75-80% and even still more preferably atleast about 85-95%.

In an even more preferred embodiment, the present invention provides anisolated nucleic acid molecule which is at least 40% identical to thenucleotide sequence set forth in SEQ ID NO:1 or SEQ ID NO:3 or to acomplementary strand thereof or a homologue, analogue or derivativethereof and is capable of hybridising under at least low stringencyconditions to a nucleotide sequence set forth in SEQ ID NO:1 or SEQ IDNO:3.

In a particularly preferred embodiment, the isolated nucleic acidmolecule described herein is further capable of encoding a sequence ofamino acids which is capable of carrying out the enzyme reactioncatalysed by a α-N-acetylglucosaminidase enzyme.

The isolated nucleic acid molecule of the present invention is alsouseful for developing a genetic construct comprising a sense molecule,for the expression or over-expression of α-N-acetylglucosaminidase inprokaryotic or eukaryotic cells. Particularly preferred eukaryotic cellsinclude CHO cells but may be in any other suitable mammalian cells orcell lines or non-mammalian cells such as yeast or insect cells.

The term “sense molecule” as used herein shall be taken to refer to anisolated nucleic acid molecule of the invention as described herein,which is provided in a format suitable for its expression to produce arecombinant polypeptide, when said sense molecule is introduced into ahost cell.

In a particularly preferred embodiment, a sense molecule which encodesthe α-N-acetylglucosaminidase comprises a sequence of nucleotides setforth in SEQ ID NO:1 or SEQ ID NO:3 or a complementary strand,homologue, analogue or derivative thereof.

In a most particularly preferred embodiment, the sense molecule of theinvention comprises the sequence of nucleotides set forth in SEQ ID NO:1or a complementary strand, homologue, analogue or derivative thereof.

Those skilled in the art will be aware that expression of a sensemolecule may require the nucleic acid molecule of the invention to beplaced in operable connection with a promoter sequence to produce a“sense construct”. The choice of promoter for the present purpose mayvary depending upon the level of expression of the sense moleculerequired and/or the tissue-specificity or developmental-specificity ofexpression of the sense molecule which is required. The sense constructmay further comprise a terminator sequence and be introduced into asuitable host cell where it is capable of being expressed to produce arecombinant polypeptide gene product.

In the context of the present invention, a sense molecule whichcorresponds to a genetic sequence or isolated nucleic acid moleculewhich encodes α-N-acetylglucosaminidase polypeptide or a homologue,analogue or derivative thereof, placed operably under the control of asuitable promoter sequence, is introduced into a cell using any suitablemethod for the transformation of said cell and said genetic sequence orisolated nucleic acid molecule is expressed therein to produce saidpolypeptide.

The present invention clearly extends to genetic constructs designed tofacilitate expression of any nucleic acid molecule described herein.

A genetic construct of the present invention comprises the foregoingsense molecule, placed operably under the control of a promoter sequencecapable of regulating the expression of the said nucleic acid moleculein a prokaryotic or eukaryotic cell, preferably a mammalian cell such asa CHO cell, a yeast cell, insect cell or bacterial cell. The saidgenetic construct optionally comprises, in addition to a promoter andsense molecule, a terminator sequence.

The term “terminator” refers to a DNA sequence at the end of atranscriptional unit which signals termination of transcription.Terminators are 3′-non-translated DNA sequences containing apolyadenylation signal, which facilitates the addition of polyadenylatesequences to the 3′-end of a primary transcript. Terminators active inplant cells are known and described in the literature. They may beisolated from bacteria, fungi, viruses, animals and/or plants.

Reference herein to a “promoter” is to be taken in its broadest contextand includes the transcriptional regulatory sequences of a classicalgenomic gene, including the TATA box which is required for accuratetranscription initiation, with or without a CCAAT box sequence andadditional regulatory elements (i.e. upstream activating sequences,enhancers and silencers) which alter gene expression in response todevelopmental and/or external stimuli, or in a tissue-specific manner. Apromoter is usually, but not necessarily, positioned upstream or 5′, ofa structural gene, the expression of which it regulates. Furthermore,the regulatory elements comprising a promoter are usually positionedwithin 2 kb of the start site of transcription of the gene.

In the present context, the term “promoter” is also used to describe asynthetic or fusion molecule, or derivative which confers, activates orenhances expression of said sense molecule in a cell.

Preferred promoters may contain additional copies of one or morespecific regulatory elements, to further enhance expression of the sensemolecule and/or to alter the spatial expression and/or temporalexpression of said sense molecule. For example, regulatory elementswhich confer copper inducibility may be placed adjacent to aheterologous promoter sequence driving expression of a sense molecule,thereby conferring copper inducibility on the expression of saidmolecule.

Placing a sense molecule under the regulatory control of a promotersequence means positioning the said molecule such that expression iscontrolled by the promoter sequence. Promoters are generally positioned5′ (upstream) to the genes that they control. In the construction ofheterologous promoter/structural gene combinations it is generallypreferred to position the promoter at a distance from the genetranscription start site that is approximately the same as the distancebetween that promoter and the gene it controls in its natural setting,i.e., the gene from which the promoter is derived. As is known in theart, some variation in this distance can be accommodated without loss ofpromoter function. Similarly, the preferred positioning of a regulatorysequence element with respect to a heterologous gene to be placed underits control is defined by the positioning of the element in its naturalsetting, i.e., the genes from which it is derived. Again, as is known inthe art, some variation in this distance can also occur.

Examples of promoters suitable for use in genetic constructs of thepresent invention include viral, fungal, bacterial, animal and plantderived promoters capable of functioning in animal, human, yeast, insector bacterial cells. The promoter may regulate the expression of the saidmolecule constitutively, or differentially with respect to the tissue inwhich expression occurs or, with respect to the developmental stage atwhich expression occurs, or in response to external stimuli such asphysiological stresses, or plant pathogens, or metal ions, amongstothers. Preferably, the promoter is capable of regulating expression ofa sense molecule in a cell derived from an animal species or human.

In a particularly preferred embodiment, the promoter is derived from thegenomic gene encoding α-N-acetylglucosaminidase, preferably the humanα-N-acetylglucosaminidase gene. In a more preferred embodiment, however,the promoter is derived from the nucleotide sequence set forth in SEQ IDNO:3 or is at least capable of hybridising to nucleotide residues 1 to989 of SEQ ID NO:3 or at least 20 contiguous nucleotides derivedtherefrom.

In an even more particularly preferred embodiment, the promoter is theCMV promoter sequence or a promoter sequence derived therefrom.

An alternative embodiment of the invention is directed to a geneticconstruct comprising a promoter or functional derivative, part fragment,homologue, or analogue thereof, derived from theα-N-acetylglucosaminidase genomic gene defined by SEQ ID NO:3.

Preferably, said genetic construct further comprises theα-N-acetylglucosaminidase sequence defined by SEQ ID NO:1 placed inoperably connection with said promoter.

A further aspect of the present invention is directed to syntheticα-N-acetylglucosaminidase or like molecule.

The term “synthetic” as used herein shall be taken to include bothrecombinant and chemically-synthesised molecules produced by thesequential addition of amino acid residues or groups of amino acidresidues in defined order.

In one embodiment, the invention relates to recombinantα-N-acetylglucosaminidase or like molecule encoded by or expressed fromthe nucleic acid molecules as hereinbefore described.

In another embodiment, the synthetic α-N-acetylglucosaminidase or likemolecule comprises a sequence of amino acids which is at least 40%identical to the amino acid sequence set forth in any one of SEQ IDNos:2, 4, 5 or 6.

More preferably, the percentage identity is at least 60% and still morepreferably at least 80% or 85-90%.

A particularly preferred embodiment of the present invention provides asynthetic α-N-acetylglucosaminidase as hereinbefore defined whichcomprises a sequence of amino acids substantially as set forth in anyone of SEQ ID Nos:2, 4, 5 or 6 or a homologue, analogue or derivativethereof.

For the purposes of nomenclature, the amino acid sequence set forth inSEQ ID NO:2 comprises the full-length translation product of the humanα-N-acetylglucosaminidase gene (i.e. hereinafter referred to as the“α-N-acetylglucosaminidase polypeptide” or “SEQ ID NO:2”) produced byexpression of either the cDNA sequence defined by SEQ ID NO:1 or thegenomic gene defined by SEQ ID NO:3. The α-N-acetylglucosaminidasepolypeptide comprises at least seven potentially-glycosylated Asnresidues, at positions 261, 272, 435, 503, 513, 526 and 532.Furthermore, the amino acid sequence of the α-N-acetylglucosaminidasepolypeptide may comprise a signal peptide of approximately 23 amino acidresidues in length, with a probable site for signal peptide peptidasecleavage occurring between Gly₂₃ and Asp₂₄.

The amino acid sequences set forth in SEQ ID Nos:4-6 relate toN-terminal and internal (i.e. CNBr) amino acid sequences derived fromhuman α-N-acetylglucosaminidase, purified as described in Example 1. Asdescribed in Example 2, the purified form of the enzyme comprises twopolypeptides having approximate molecular weights of 82 and 77 kDa. Thesequence set forth in SEQ ID NO:4 relates to the N-terminal sequence ofthe 82 kDa polypeptide, while SEQ ID NO:5 relates to the N-terminalsequence of the 77% kDa polypeptide. Furthermore, SEQ ID NO:4 comprisesamino acids residues 24-43 of SEQ ID NO:2, while SEQ ID NO:5 comprisesamino acid residues 59-76 of SEQ ID NO:2.

The amino acid sequence defined by SEQ ID NO:6 relates to theCNBr-cleaved peptide of purified human α-N-acetylglucosaminidase. Thisamino acid sequence aligns with amino acid residues 540-554 of theα-N-acetylglucosaminidase polypeptide (SEQ ID NO:2).

In the present context, “homologues” of a polypeptide refer to thosepolypeptides, enzymes or proteins which have a similarα-N-acetylglucosaminidase enzyme activity, notwithstanding any aminoacid substitutions, additions or deletions thereto. A homologue may beisolated or derived from the same or another animal species.

Furthermore, the amino acids of a homologous polypeptide may be replacedby other amino acids having similar properties, for examplehydrophobicity, hydrophilicity, hydrophobic moment, charge orantigenicity, and so on.

“Analogues” encompass α-N-acetylglucosaminidase polypeptides and peptidederivatives thereof notwithstanding the occurrence of any non-naturallyoccurring amino acid analogues therein.

The term “derivative” in relation to the polypeptides of the inventionrefer to mutants, parts or fragments of a functional molecule.Derivatives include modified peptides in which ligands are attached toone or more of the amino acid residues contained therein, such ascarbohydrates, enzymes, proteins, polypeptides or reporter moleculessuch as radionuclides or fluorescent compounds. Glycosylated,fluorescent, acylated or alkylated forms of the subject peptides areparticularly contemplated by the present invention. Additionally,derivatives of a polypeptide may comprise fragments or parts of an aminoacid sequence disclosed herein and are within the scope of theinvention, as are homopolymers or heteropolymers comprising two or morecopies of the subject polypeptides. Procedures for derivatizing peptidesare well-known in the art.

Accordingly, this aspect of the present invention is directed to anyproteinaceous molecule comprising an amino acid sequence correspondingto the full length mammalian α-N-acetylglucosaminidase enzyme or to alike molecule. The like molecule, therefore, comprises parts,derivatives and/or portions of the α-N-acetylglucosaminidase enzymewhether functional or not.

Preferably, the mammal is human but may be of non-human origin ascontemplated above.

The synthetic or recombinant α-N-acetylglucosaminidase of the presentinvention may comprise an amino acid sequence corresponding to thenaturally occurring amino acid sequence or may contain single ormultiple amino acid substitutions, deletions and/or additions. Thelength of the amino acid sequence may range from a few residues to afull length molecule.

Amino acid substitutions are typically of single residues. Amino acidinsertions will usually be in the order of about 1-10 amino acidresidues and deletions will range from about 1-20 residues. Preferably,deletions or insertions are made in adjacent pairs, i.e. a deletion oftwo residues or insertion of two residues.

Amino acid insertional derivatives of α-N-acetylglucosaminidase of thepresent invention include amino and/or carboxyl terminal fusions as wellas intra-sequence insertions of single or multiple amino acids.Insertional amino acid sequence variants are those in which one or moreamino acid residues are introduced into a predetermined site in theprotein although random insertion is also possible with suitablescreening of the resulting product. Deletional variants arecharacterised by the removal of one or more amino acids from thesequence. Substitutional amino acid variants are those in which at leastone residue in the sequence has been removed and a different residueinserted in its place. Typical substitutions are those made inaccordance with the following Table 2:

The amino acid variants referred to above may readily be made usingpeptide synthetic techniques well known in the art, such as solid phasepeptide synthesis (Merrifield synthesis) and the like, or by recombinantDNA manipulations. Techniques for making substitution mutations atpredetermined sites in DNA having known or partially known sequence arewell known and include, for example, M13 mutagenesis. The manipulationof DNA sequence to produce variant proteins which manifest assubstitutional, insertional or deletional variants are convenientlyelsewhere described such as Sambrook et al, 1989 Molecular Cloning: ALaboratory Manual Cold Spring Harbor Laboratories, Cold Spring Harbor,N.Y.

The derivatives or like molecules include single or multiplesubstitutions, deletions and/or additions of any component(s) naturallyor artificially associated with the α-N-acetylglucosaminidase enzymesuch as carbohydrate, lipid and/or other proteinaceous moieties. Forexample, the present invention extends to glycosylated andnon-glycosylated forms of the molecule. All such molecules areencompassed by the expression “mutants”, “derivatives”, “fragments”,“portions” and “like” molecules. These molecules may be active ornon-active and may contain specific regions, such as a catalytic region.Particularly, preferred derivative molecules include those with alteredglycosylation patterns relative to the naturally occurring molecule.Even more particularly, the recombinant molecule is more highlyglycosylated than the naturally occurring molecule. Such highlyglycosylated derivatives may have improved take-up properties andenhanced half-lives.

As indicated in the Examples, the molecular weight of purified humanα-N-acetylglucosaminidase (i.e. 82 kDa and 77 kDa) and recombinantmammalian α-N-acetylglucosaminidase produced in CHO cells (i.e. 89 kDaand 79 kDa) are greater than the deduced molecular weight of theα-N-acetylglucosaminidase polypeptide set forth in SEQ ID No:2 (i.e. 70kDa), suggesting that the purified and recombinant polypeptide arepost-translationally modified. The data presented in Example 8 indicatefurther that the recombinant α-N-acetylglucosaminidase enzyme producedin CHO cells, at least, is glycosylated and that the difference inmolecular weight determined for the recombinant polypeptides and thepolypeptide of SEQ ID No:2 is due almost entirely to glycosylation ofthe recombinant polypeptide by CHO cells. As shown in Example 9, theglycosylated recombinant α-N-acetylglucosaminidase polypeptide exhibitsenzymatic activity.

The present invention also extends to syntheticα-N-acetylglucosaminidase or like molecules when fused to otherproteinaceous molecules. The latter may include another enzyme, reportermolecule, purification site or an amino acid sequence which facilitatestransport of the molecule out of a cell, such as a signal sequence.

The present invention extends further to post-translationalmodifications to the α-N-acetylglucosaminidase enzyme. The modificationsmay be made to the naturally occurring enzyme or following synthesis byrecombinant techniques. The modifications may be at the structural levelor at, for example, the electrochemical level such as modifying netcharge or structural conformation of the enzyme.

Such modification may be important to facilitate entry or penetration ofthe enzyme into selected tissues such as cartilage or blood brainbarriers or to increase circulation half-life.

Analogues of α-N-acetylglucosaminidase contemplated herein include, butare not limited to, modifications to side chains, incorporation ofunnatural amino acids and/or their derivatives during peptide synthesisand the use of crosslinkers and other methods which imposeconformational constraints on the enzyme.

Examples of side chain modifications contemplated by the presentinvention include modifications of amino groups such as by reductivealkylation by reaction with an aldehyde followed by reduction withNaBH₄; amidination with methylacetimidate; acylation with aceticanhydride; carbamoylation of amino groups with cyanate;trinitrobenzylation of amino groups with 2,4,6-trinitrobenzene sulphonicacid (TNBS); acylation of amino groups with succinic anhydride andtetrahydrophthalic anhydride; and pyridoxylation of lysine withpyridoxal-5′-phosphate followed by reduction with NaBH₄.

The guanidino group of arginine residues may be modified by theformation of heterocyclic condensation products with reagents such as2,3-butanedione, phenylglyoxal and glyoxal.

The carboxyl group may be modified by carbodiimide activation viaO-acylisourea formation followed by subsequent derivatisation, forexample, to a corresponding amide.

Sulphydryl groups may be modified by methods such as carboxymethylationwith iodoacetic acid or iodoacetamide; performic acid oxidation tocysteic acid; formation of a mixed disulphides with other thiolcompounds; reaction with maleimide, maleic anhydride or othersubstituted maleimide; formation of mercurial derivatives using4-chloromercuribenzoate, 4-chloromercuriphenylsulphonic acid,phenylmercury chloride, 2-chloromercuric-4-nitrophenol and othermercurials; carbamoylation with cyanate at alkaline pH.

Tryptophan residues may be modified by, for example, oxidation withN-bromosuccinimide or alkylation of the indole ring with2-hydroxy-5-nitrobenzyl bromide or sulphenyl halides. Tyrosine residueson the other hand, may be altered by nitration with tetranitromethane toform a 3-nitrotyrosine derivative.

Modification of the imidazole ring of a histidine residue may beaccomplished by alkylation with iodoacetic acid derivatives orN-carbethoxylation with diethylpyrocarbonate.

Examples of incorporating unnatural amino acids and derivatives duringpeptide synthesis include, but are not limited to, use of norleucine,4-amino butyric acid, 4-amino-3-hydroxy-5-phenylpentanoic acid,6-aminohexanoic acid, t-butylglycine, norvaline, phenylglycine,ornithine, sarcosine, 4-amino-3-hydroxy-6-methylheptanoic acid,2-thienyl alanine and/or D-isomers of amino acids. Non-naturallyoccurring amino acids contemplated by the present invention areincorporated herein, as Table 3.

Crosslinkers can be used, for example, to stabilise 3D conformations,using homo-bifunctional crosslinkers such as the bifunctional imidoesters having (CH₂)_(n) spacer groups with n=1 to n=6, glutaraldehyde,N-hydroxysuccinimide esters and hetero-bifunctional reagents whichusually contain an amino-reactive moiety such as N-hydroxysuccinimideand another group specific-reactive moiety such as maleimido or dithiomoiety (SH) or carbodiimide (COOH). In addition, the enzyme could beconformationally constrained by, for example, incorporation of C_(α) andN_(α)-methylamino acids, introduction of double bonds between C_(α) andC_(β) atoms of amino acids and the formation of cyclic peptides oranalogues by introducing covalent bonds such as forming an amide bondbetween the N and C termini, between two side chains or between a sidechain and the N or C terminus.

Electrochemical modifications of α-N-acetylglucosaminidase includeinteraction with polylysine or polyethylene glycol or other agent whicheffects an overall change to the net charge of the enzyme.

Advantageously, the recombinant α-N-acetylglucosaminidase is abiologically pure preparation meaning that it has undergone somepurification away for other proteins and/or non-proteinaceous material.The purity of the preparation may be represented as at least 40% of theenzyme, preferably at least 60%, more preferably at least 75%, even morepreferably at least 85% and still more preferably at least 95% relativeto non-α-N-acetylglucosaminidase material as determined by weight,activity, amino acid homology or similarity, antibody reactivity orother convenient means.

Particularly preferred methods for the preparation and purification ofrecombinant α-N-acetylglucosaminidase are provided in Examples 7 and 8.

Those skilled in the art will be aware of the means of purifying asynthetic or recombinant α-N-acetylglucosaminidase from several sourceswithout undue experimentation and for expressing the degree of purity ofsuch a purified preparation of the enzyme.

The present invention further contemplates antibodies toα-N-acetylglucosaminidase and preferably syntheticα-N-acetylglucosaminidase or like molecule. The antibodies may bepolyclonal or monoclonal, naturally occurring or synthetic (includingrecombinant, fragment or fusion forms). Such antibodies will be usefulin developing immunoassays for α-N-acetylglucosaminidase and foridentifying additional genetic sequences which are capable of expressingα-N-acetylglucosaminidase polypeptides or homologues, analogues orderivatives thereof.

Both polyclonal and monoclonal antibodies are obtainable by immunisationwith an appropriate synthetic or recombinant gene product, or epitope,or peptide fragment of a gene product, in particular aα-N-acetylglucosaminidase polypeptide or a homologue, analogue orderivative thereof.

Alternatively, fragments of antibodies may be used, such as Fabfragments. The present invention extends further to encompassrecombinant and synthetic antibodies and to antibody hybrids. A“synthetic antibody” is considered herein to include fragments andhybrids of antibodies.

A further aspect of the present invention contemplates a method ofscreening for mutations or other abberations in theα-N-acetylglucosaminidase gene in a human or animal patient. Such amethod may be accomplished in a number of ways including isolating asource of DNA to be tested or mRNA therefrom and hybridising thereto anucleic acid molecule as hereinbefore described. Generally, the nucleicacid is probe or primer size and polymerase chain reaction is aconvenient means by which to analyse the RNA or DNA. Other suitableassays include the ligation chain reaction and the strand displacementamplification methods. The α-N-acetylglucosaminidase sequence can alsobe determined and compared to the naturally occurring sequence. Suchmethods may be useful in adults and children and may be adapted for apre-natal test. The DNA to be tested includes a genomic sample carryingthe α-N-acetylglucosaminidase gene, a cDNA clone and/or amplificationproduct.

In accordance with this aspect of the present invention there isprovided a method for screening for abberations in theα-N-acetylglucosaminidase gene including the absence of such a gene or aportion or a substantial portion thereof comprising isolating a sampleof DNA or mRNA corresponding to a region of said DNA and contacting samewith an oligonucleotide probe capable of hybridising to one or morecomplementary sequences within the α-N-acetylglucosaminidase gene andthen detecting the hybridisation, the extent of hybridisation or theabsence of hybridisation.

Alternatively, the probe is a primer and capable of directingamplification of one or more regions of said α-N-acetylglucosaminidasegene and the amplification products and/or profile of amplificationproducts is compared to an individual carrying the full gene or to areference date base.

Conveniently, the amplification products are sequenced to determine thepresence or absence of the full gene.

The present invention extends to the use of any and all DNA-based ornucleic acid-based hybridisation and/or polymerase chain reactionformats as described herein, for the diagnosis of a disorder involvingthe α-N-acetylglucosaminidase gene in a human or animal patient.

The present invention further extends to a method of treating patientssuffering from α-N-acetylglucosaminidase deficiency, such as inMPS-IIIB, said method comprising administering to said patient aneffective amount of α-N-acetylglucosaminidase or active like formthereof.

Preferably, the α-N-acetylglucosaminidase is in recombinant form. Such amethod is referred to as “enzyme therapy”. Alternatively, gene therapycan be employed including introducing an active gene (i.e. a nucleicacid molecule as hereinbefore described) or to parts of the gene orother sequences which facilitate expression of a naturally occurringα-N-acetylglucosaminidase gene.

Administration of α-N-acetylglucosaminidase for enzyme therapy may be byoral, intravenous, suppository, intraperitoneal, intramuscular,intranasal, intradermal or subcutaneous administration or by infusion orimplantation. The α-N-acetylglucosaminidase is preferably ashereinbefore described including active mutants or derivatives thereofand glycosylation variants thereof. Administration may also be by way ofgene therapy including expression of the gene by inclusion of the genein viral vectors which are introduced into the animal (e.g. human) hostto be treated. Alternatively, the gene may be expressed in a bacterialhost which is then introduced and becomes part of the bacterial flora inthe animal to be tested.

Still yet another aspect of the present invention is directed to apharmaceutical composition comprising synthetic (e.g. recombinant)α-N-acetylglucosaminidase or like molecule, including active derivativesand fragments thereof, alone or in combination with other activemolecules. Such other molecules may act synergistically with the enzymeor facilitates its entry to a target cell. The composition will alsocontain one or more pharmaceutically acceptable carriers and/ordiluents. The composition may alternatively comprise a genetic componentuseful in gene therapy.

The active ingredients of the pharmaceutical composition comprising thesynthetic or recombinant α-N-acetylglucosaminidase or mutants orfragments or derivatives thereof are contemplated to exhibit excellentactivity in treating patients with a deficiency in the enzyme whenadministered in an amount which depends on the particular case. Thevariation depends, for example, on the patient and theα-N-acetylglucosaminidase used. For example, from about 0.5 ug to about20 mg of enzyme per animal body or, depending on the animal and otherfactors, per kilogram of body weight may be administered. Dosage regimamay be adjusted to provide the optimum therapeutic response. Forexample, several divided doses may be administered daily, weekly,monthly or in other suitable time intervals or the dose may beproportionally reduced as indicated by the exigencies of the situation.Accordingly, alternative dosages in the order of 1.0 μg to 15 mg, 2.0 μgto 10 mg or 10 μg to 5 mg may be administered in a single or as part ofmultiple doses. The active compound may be administered in a convenientmanner such as by the oral, intravenous (where water soluble),intramuscular, subcutaneous, intranasal, intradermal or suppositoryroutes or implanting (eg using slow release molecules). Depending on theroute of administration, the active ingredients which comprise asynthetic (e.g. recombinant) α-N-acetylglucosaminidase or fragments,derivatives or mutants thereof may be required to be coated in amaterial to protect same from the action of enzymes, acids and othernatural conditions which may inactivate said ingredients. For example,the low lipophilicity of α-N-acetylglucosaminidase will allow it to bedestroyed in the gastrointestinal tract by enzymes capable of cleavingpeptide bonds and in the stomach by acid hydrolysis. In order toadminister the vaccine by other than parenteral administration, theenzyme will be coated by, or administered with, a material to preventits inactivation. For example, the enzyme may be administered in anadjuvant, co-administered with enzyme inhibitors or in liposomes.Adjuvant is used in its broadest sense and includes any immunestimulating compound such as interferon. Adjuvants contemplated hereininclude resorcinols, non-ionic surfactants such as polyoxyethylene oleylether and n-hexadecyl polyethylene ether. Conveniently, the adjuvant isFreund's Complete or Incomplete Adjuvant. Enzyme inhibitors includepancreatic trypsin inhibitor, diisopropylfluorophosphate (DEP) andtrasylol. Liposomes include water-in-oil-in-water emulsions as well asconventional liposomes.

The active compound may also be administered in dispersions prepared inglycerol, liquid polyethylene glycols, and/or mixtures thereof and inoils. Under ordinary conditions of storage and use, these preparationscontain a preservative to prevent the growth of microorganisms.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. In all cases the form must be sterile and mustbe fluid to the extent that easy syringability exists. It must be stableunder the conditions of manufacture and storage and must be preservedagainst the contaminating action of microorganisms such as bacteria andfungi. The carrier can be a solvent or dispersion medium containing, forexample, water, ethanol, polyol (for example, glycerol, propyleneglycol, and liquid polyethylene glycol, and the like), suitable mixturesthereof, and vegetable oils. The proper fluidity can be maintained, forexample, by the use of a coating such as lecithin, by the maintenance ofthe required particle size in the case of dispersion and by the use ofsuperfactants. The prevention of the action of microorganisms can bebrought about by various antibacterial and antifungal agents, forexample, parabens, chlorobutanol, phenol, sorbic acid, thirmerosal, andthe like. In many cases, it will be preferable to include isotonicagents, for example, sugars or sodium chloride. Prolonged absorption ofthe injectable compositions can be brought about by the use in thecompositions of agents delaying absorption, for example, aluminummonostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the activecompound in the required amount in the appropriate solvent with variousof the other ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the various sterilized active ingredient(s) into a sterilevehicle which contains the basic dispersion medium and the requiredother ingredients from those enumerated above. In the case of sterilepowders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum drying and the freeze-dryingtechnique which yield a powder of the active ingredient plus anyadditional desired ingredient from previously sterile-filtered solutionthereof.

When the α-N-acetylglucosaminidase of the present invention is suitablyprotected as described above, the composition may be orallyadministered, for example, with an inert diluent or with an assimilableedible carrier, or it may be enclosed in hard or soft shell gelatincapsule, or it may be compressed into tablets, or it may be incorporateddirectly with the food of the diet. For oral therapeutic administration,the active compound may be incorporated with excipients and used in theform of ingestible tablets, buccal tablets, troches, capsules, elixirs,suspensions, syrups, wafers, and the like. Such compositions andpreparations should contain at least 1% by weight of active compound.The percentage of the compositions and preparations may, of course, bevaried and may conveniently be between about 5 to about 80% of theweight of the unit. The amount of active compound in the vaccinecompositions is such that a suitable dosage will be obtained. Preferredcompositions or preparations according to the present invention areprepared, so that an oral dosage unit form contains between about 0.5 ugand 20 mg of active compound.

The tablets, troches, pills, capsules and the like may also contain thefollowing: a binder such as gum gragacanth, acacia, corn starch orgelatin; excipients such as dicalcium phosphate; a disintegrating agentsuch as corn starch, potato starch, alginic acid and the like; alubricant such as magnesium stearate; and a sweetening agent such asucrose, lactose or saccharin may be added or a flavoring agent such aspeppermint, oil of wintergreen, or cherry flavouring. When the dosageunit form is a capsule, it may contain, in addition to materials of theabove type, a liquid carrier. Various other materials may be present ascoatings or to otherwise modify the physical form of the dosage unit.For instance, tablets, pills, or capsules may be coated with shellac,sugar or both. A syrup or elixir may contain the active compound,sucrose as a sweetening agent, methyl and propylparabens aspreservatives, a dye and flavoring such as cherry or orange flavor. Ofcourse, any material used in preparing any dosage unit form should bepharmaceutically pure and substantially non-toxic in the amountsemployed. In addition, the active compound may be incorporated intosustained-release reparations and formulations.

As used herein “pharmaceutically acceptable carriers and/or diluents”include any and all solvents, dispersion media, aqueous solutions,coatings, antibacterial and antifungal agents, isotonic and absorptiondelaying agents, and the like. The use of such media and agents forpharmaceutical active substances is well known in the art. Exceptinsofar as any conventional media or agent is incompatible with theactive ingredient, use thereof in the pharmaceutical compositions iscontemplated. Supplementary active ingredients can also be incorporatedinto the compositions.

The present invention further relates to the use ofα-N-acetylglucosaminidase or active fragment, mutant or derivativethereof in the manufacture of a medicament for the treatment of patientssuffering from a deficiency in the naturally occurring enzyme (e.g.MPS-IIIB).

The present invention is further described with reference to thefollowing non-limiting Examples.

EXAMPLE 1 Purification of α-N-Acetylglucosaminidase

α-N-acetylglucosaminidase was purified according to the method describedin Weber et al. (1996). Enzyme was purified to homogeneity from humanplacenta. Evidence of purity is shown following SDS/PAGE which isrepresented in FIG. 1. All samples were reduced with dithiothreitolprior to electrophoresis.

EXAMPLE 2 Characterisation of α-N-Acetylglucosaminidase

Results presented in FIG. 1 show two polypeptides of about 82 kDa and77% kDa molecular weight, which correspond to α-N-acetylglucosaminidasepolypeptides purified from human placenta according to Example 1.

EXAMPLE 3 Amino Acid Sequence Determination

The N-terminal amino acid sequences the 77% kDA and 82 kDaα-N-acetylglucosaminidase polypeptides, in addition to the amino acidsequence of an internal CNBr cleavage product of these peptides, weredetermined using the methods of Weber et al. (1996).

The amino acid sequences are shown in Table 4.

TABLE 4 N-Terminal amino acid sequences (SEQ ID NO:4 and SEQ ID No:5)and CNBr peptide sequence (SEQ ID No:6) determined from purified humanα-N-Acetylglucosaminidase polypeptide 82 kDa DEAREAAAVRALVARLLGPGpolypeptide 77 kDa KPGLDTYSLGGGGAAX¹ VR CNBr peptide WRLLLTSAPSLX¹ TX¹PX¹ no residue could be identified for this position, indicating thatthis residue could be phosphorylated or-glycosylated.

EXAMPLE 4 Cloning of α-N-Acetylglucosaminidase cDNA

Oligonucleotide probes were prepared based on the partial amino acidsequences obtained for the purified α-N-acetylglucosaminidasepolypeptides (Example 3). The probes were subsequently used to screen ahuman peripheral blood leukocyte cDNA library. An approximately 2.6 kbpcDNA clone was isolated encoding most of the sequence of humanα-N-acetylglucosaminidase (SEQ ID NO:1).

The remaining α-N-acetylglucosaminidase coding sequence was obtainedfrom the nucleotide sequence of the corresponding genomic gene (SEQ IDNO:3), isolated by hybridisation to a human chromosome 17 library (Weberet. al. 1996).

The complete open reading frame is 2232 nucleotides long and encodes a743 (plus stop codon) amino acid protein. The predicted molecular massof the longest mature protein (minus the 23 amino acid N-terminal signalpeptide) is about 79,622 daltons.

The amino acid sequence of α-N-acetylglucosaminidase is shown in SEQ IDNO:2. The deduced molecular weight of the desired amino acid sequence ofα-N-acetylglucosaminidase is approximately 70 kDa. The probable site ofsignal peptide peptidase cleavage is between amino acids 23 and 24.There are seven potential N-glycosylation sites in the sequence.

The nucleotide sequence of the corresponding α-N-acetylglucosaminidasegenomic gene (SEQ ID No:3) comprises 10380 bp including 889 bp of 5′upstream sequence corresponding to at least at part of theα-N-acetylglucosaminidase promoter sequence, in addition to thenucleotide sequences of introns I, II, II, IV, V, in addition to 1326 bpof 3′-untranslated sequence.

EXAMPLE 5 Construction of an Expression Vector Comprising theα-N-Acetylglucosaminidase cDNA Sequence

The cDNA insert of λ clone pbl 33, containing bases 107 to 2575 of theα-N-acetylglucosaminidase cDNA was excised with EcoRI and subcloned intopBluescript II SK-(Stratagene). A 178 bp XmaI fragment (bases 1 to 178of the α-N-acetylglucosaminidase cDNA) from cosmid sub-clone 6.3,containing the start codon, was cloned into the pBluescript subclone toproduce a full-length cDNA sequence in addition to 101 bp of 5′non-translated sequence as well as 245 bp of 3′ non-translated regionincluding the polyadenylation-site, the poly A-tail and linkerDNA. Thefull length cDNA was directionally cloned into the pCDNA3expressionvector (Invitrogen) via the EcoRI and BamHI sites.

EXAMPLE 6 Expression of Recombinant α-N-acetylglucosaminidase

Chinese Hamster Ovary (CHO) cells were transfected with expressionvectorusing the DOTAP transfection reagent (Boehringer Mannheim) according tothe manufacturers instructions. Cells were grown in Ham's F12 medium,10% (v/v) fetal calf serum, penicillin and streptomycin sulfate at 100μg/ml each. Cells were grown in nonselective medium for 48 h and thenincubated in medium containing 750 μg/ml G418 sulfate (Geniticin) untilresistant colonies emerged.

Single cell clones were grown up and 26 of them were tested forexpression of recombinant α-N-acetylglucosaminidase with a fluorogenicα-N-acetylglucosaminidase substrate. (i.e. N-acetylglucosamine α-linkedto 4-methylumbelliferone)

EXAMPLE 7 Large Scale α-N-acetylglucosaminidase Production

2 g of Cytodex 2 microcarrier beads were swollen in 250 ml of PBS for 3h at 37° C. with three changes of PBS and then autoclaved for 15 min at120° C. (wet cycle). The beads were then rinsed with sterile growthmedium (Coons/DMEM, 10% v/v fetal calf serum, penicillin andstreptomycin sulfate at 100 μg/ml each and 0.1% w/v Pluronic F68) andtransferred into a Techne stirrer culture flask. The microcarrier beadswere inoculated with seven confluent 175 flasks of the cell cloneshowing the highest expression of recombinant α-N-acetylglucosaminidase.Growth medium was added up to 200 ml and the culture incubated with astirrer speed of 20 rpm to achieve an even distribution of cells on thebeads. The cells were allowed to attach to the beads for 16 h at lowspeed then medium was added up to 500 ml and the stirrer speed increasedto 30 rpm. After a growth phase of 48 to 72 h with daily aerating toallow gas exchange the beads were completely covered with cells and themedium was exchanged for production medium (Coons/DMEM, no fetal calfserum, penicillin and streptomycin sulfate at 100 μg/ml each, 0.1% w/vPluronic F68 and 5 mM NH₄Cl). The glucose concentration was monitoreddaily and the medium replaced, when glucose fell below 5 mM every 203days. The harvested medium contained approximately 2 mgα-N-acetylglucosaminidase protein per dm³ of production medium.

EXAMPLE 8 Purification of Recombinant α-N-acetylglucosaminidase

Production medium was dialysed against 50 mM NaAc pH 5.5 and loaded ontoa heparin-agarose column equilibrated in the same buffer. After washingwith NaAc buffer and NaAc/50 mM NaCl the column was eluted with 75 mMNaCl in NaAc buffer. The eluate was dialysed against 20 mM Tris/HCl pH7.5, loaded onto a DEAE Scphacel column, washed with 25 mM NaCl in 20 mMTris/HCl and then eluted with 50 and 75 mM NaCl in 20 mM Tris/HClrespectively.

SDS-PAGE of the two eluates showed two polypeptide bands associated withenzyme activity with apparent molecular weights of 79 and 89 kDa. Thesmaller α-N-acetylglucosaminidase was eluted predominantly in the 50 mMNaCl fraction whereas the 89 kDa α-N-acetylglucosaminidase polypeptidewas enriched in the 75 mM NaCl fraction (FIG. 2).

The difference in apparent molecular weight of the recombinantα-N-acetylglucosaminidase polypeptides is due to the presence ofadditional carbohydrate side chains, since a digest with PNGase F, whichcleaves off N-glycosylation groups, reduced both the 79 kDa and 89 kDapolypeptides to the polypeptide band having an apparent molecular weightof about 70 kDa (FIG. 2), which corresponds to the approximate molecularweight deduced from primary amino acid sequence data (SEQ ID No:2).

EXAMPLE 9 Characteristics of Recombinant α-N-acetylglucosaminidase

No differences were observed between the enzyme activities of the 79 and89 kDa recombinant α-N-acetylglucosaminidase polypeptides produced inCHO cells according to Example 7 and 8. With the fluorogenicN-acetylglucosamine α-linked to 4-methylumbelliferone (4-MU) substrate,the enzyme has a pH-optimum of 4.6 with a k_(M) of 5.34 mM and a V_(max)of 3.97×10⁶ pmol/min/mg. Towards a ³H-labelled disaccharide substrate itshould a pH-optimum of 4.1 with a k_(M) of 0.0166 mM and a V_(max) of4.48×10⁴ pmol/min/mg.

EXAMPLE 10 Mutational Analysis of Sanfilippo B Patients

Genomic DNA is isolated from cultivated skin fibroblasts of patients byextraction with Phenol/Chloroform and used to amplify the eight exonsand adjacent intronic sequences individually by PC.

Primer sequences used in the amplification reaction are readilydetermined from the nucleotide sequences of theα-N-acetylglucosaminidase cDNA and genomic clones.set forth in SEQ IDNo:1 or SEQ ID No:3. Amplification conditions are also readilydetermined without undue experimentation. Procedures for the design ofPCR primers and amplification conditions are described in detail, forexample, by McPherson et al. (1991). Differences in the primary sequencecan be identified by separating the PCR products on a polyacrylamide gelunder non-denaturing conditions (SSCP gels). Base changes, insertionsand deletions will lead to a different band pattern compared with thewildtype in most of the cases, which can be visualised either byautoradiography of the gel after labelling the DNA during the PCR or bystaining unlabelled DNA in the gel with silver. PCR products which showa different band pattern are sequences to identify the change. Otherpatient samples can be tested for mutations and polymorphism that werefound by hybridisation with wildtype- and mutation-specificoligonucleotides (ASO).

Those skilled in the art will appreciate that the invention describedherein is susceptible to variations and modifications other than thosespecifically described. It is to be understood that the inventionincludes all such variations and modifications. The invention alsoincludes all of the steps, features, compositions and compounds referredto or indicated in this specification, individually or collectively, andany and all combinations of any two or more of said steps or features.

REFERENCES

-   1. Ausubel, F. M., Brent, R., Kingston, R E, Moore, D. D.,    Seidman, J. G., Smith, J. A., and Struhl, K. (1987). In: Current    Protocols in Molecular Biology. Wiley Interscience (ISBN 047150338).-   2. Hopwood J J (1989) In: “Heparin: Chemical and Biological    Properties, Clinical Applications” (Lane D W and Lindahl U, eds.),    190-229, Edward Arnold, London.-   3. McKusick V and Neufeld E (1983) In: “The Metabolic Basis of    Inherited Disease” (Stanbury J B, Wyngaarden J B, Fredrickson D S,    Goldstein J L and Brown M S, eds), 5th Ed., 751-771, McGraw-Hill,    New York.-   4. McPherson, M. J., Quirke, P. and Taylor, G. R, (1991) In: PCR A    Practical Approach. Oxford University Press, Oxford. (ISBN    0-19-96322L-X).-   5. O'Brien J S, (1972) Proc. Natl. Acad. Sci. USA 69: 1720-1722.-   6. Sambrook, J., Fritsch, E., and Maniatis, T. (1989) In: “Molecular    Cloning” a laboratory manual, Cold Spring Harbour.-   7. Von Figura, K, and Kresse H, (1972) Biochem Biophys. Res. Commun,    48: 262-269-   8. Weber B, Scott H, Blanch L, Clements P, Morris C P, Anson D,    Hopwood J, (1996) Nature Genetics (submitted)

1. A method for the production of α-N-acetylglucosaminidase, said methodcomprising: (i) transfecting mammalian cells with an expression vectorcomprising a DNA molecule encoding α-N-acetylglucosaminidase having theamino acid sequence of SEQ ID NO:2 or fragment thereof comprising aminoacids 24-743 of SEQ ID NO:2 fused to a signal sequence; (ii) culturingthe transfected cells, whereby the α-N-acetylglucosaminidase proteinhaving the amino acid sequence of SEQ ID NO:2 or fragment thereofcomprising amino acids 24-743 of SEQ ID NO:2 fused to a signal sequenceis produced and α-N-acetylglucosaminidase protein secreted into themedium; and (iii) recovering the α-N-acetylglucosaminidase protein, fromthe medium.
 2. The method according to claim 1, wherein the DNA moleculeencoding α-N-acetylglucosaminidase is the cDNA having the sequence SEQID NO:1.
 3. The method according to claim 1 wherein the mammalian cellsare CHO cells.
 4. The method according to claim 1 wherein the proteinrecovered from the medium is in a glycosylated form.
 5. The methodaccording to claim 4 wherein the molecular weight of the glycosylatedform of the protein recovered from the medium as determined usingSDS/PAGE is at least approximately 79 kDa.